DOCSLIB.ORG

I = Chapter 10. Transcription: RNA Polymerase BMB 400, Part Three Gene Expression and Protein Synthesis L

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
I = Chapter 10. Transcription: RNA Polymerase BMB 400, Part Three Gene Expression and Protein Synthesis L BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase B M B 400, Part Three Gene Expression and Protein Synthesis Lecture Notes Overview of Part Three: The pathway of gene expression Recall the Central Dogma of molecular biology: DNA is transcribed into RNA, which is translated into protein. We will cover the material in that order, since that is the direction that information flows. However, there are additional steps, in particular the primary transcript is frequently a precursor molecule that is processed into a mature RNA. E.g., the rRNA genes are transcribed into pre-rRNA, that is cleaved, methylated and modified to produce mature rRNA. Many mRNAs, especially in eukarytoes, are derived from pre-mRNAs by splicing and other processing events. This general topic will be covered after transcription and before translation. Fig. 3.1.1 BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase CHAPTER 10= PART THREE-I. TRANSCRIPTION: RNA polymerase A. RNA polymerase catalyzes the DNA-dependent synthesis of RNA 1. RNA polymerase requires DNA as a template. In duplex DNA, the template strand of DNA is copied into RNA by RNA polymerase. The choice of nucleotides during this process is directed by base complementarity, so that the sequence of RNA synthesized is the reverse complement of the DNA template strand. It is the same sequence as the nontemplate (or top) strand, except that U's are present instead of T's. This process of RNA synthesis directed by a DNA template, catalyzed by RNA polymerase, is called transcription. 2. RNA polymerase does not require a primer to initiate transcription. 3. RNA polymerase catalyzes the sequential addition of a ribonucleotide to the 3' end of a growing RNA chain, with the sequence of nucleotides specified by the template. The substrate NTP is added as a NMP with the liberation of pyrophosphate. This process occurs cyclically during the elongation phase of transcription. NTP + (NMP)n ←→ (NMP) n+1 + PPi template DNA Mg++ Fig. 3.1.2. Sequential addtion of ribonucleotides to growing RNA BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 4. The liberated pyrophosphate is cleaved in the cell to 2 Pi, an energetically favorable reaction that drives the reaction in the direction of synthesis. 5. In the presence of excess PPi, the reverse reaction of pyrophosphorolysis can occur. 6. Synthesis always proceeds in a 5' to 3' direction (with respect to the growing RNA chain). The template is read in a 3' to 5' direction. B. E. coli RNA polymerase structure 1. This one RNA polymerase synthesizes all classes of RNA mRNA, rRNA, tRNA 2. It is composed of four subunits. a. Core and holoenzyme α2ββ'σ ←→ α2ββ' + σ Holoenzyme = α2ββ'σ = core + σ = can initiate transcription accurately as the proper site, as determined by the promoter Core = α2ββ' = can elongate a growing RNA chain A promoter can be defined in two ways. (a) The sequence of DNA required for accurate, specific intiation of transcription (b) The sequence of DNA to which RNA polymerase binds to accurately initiate transcription. b. Subunits Subunit Size Gene Function β' 160 kDa rpoC β' + β form the catalytic center. β 155 kDa rpoB β' + β form the catalytic center. α 40 kDa rpoA enzyme assembly; also binds UP sequence in the promoter σ 70 kDa (general) rpoD confers specificity for promoter; binds to -10 and -35 sites in the promoter Bacteria have several σ factors, ranging in size from 32 to 92 kDa, each of which confers specificity for a different type of promoter. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase Fig. 3.1.3. Diagram of E. coli RNA polymerase α E. coli RNA polymerase α β σ β' UP -35 -10 3. Three-dimensional structure of E. coli RNA polymerase Crystals suitable for X-ray diffraction studies have not been obtained yet, but the surface topography can be determined from by electron crystallography of two-dimensional crystalline arrays. Fig. 3.1.4. Low resolution structure of RNA polymerases from electron crystallography e- Electron microscope Computer workstation Form a 2-dimensional crystal Record micrographs from the Average the information from (i.e. 1 molecule thick) on a layer of crystalline arrays at three angles the micrographs to determine a positively charged lipid. Place on (2 tilted to the incident electron low resolution map (e.g. 27 an electron microscope grid and beam and 1 untilted). Angtroms) of the surface stain with uranyl acetate. topography (i.e. the part outlined by the uranyl acetate. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase Fig. 3.1.5. E. coli RNA polymerase core (left) and holoenzyme (right) Images from analysis by Seth Darst. The structure in the presence of σ (holoenzyme) is on the right; note the open channel for DNA binding. The structure in the absence of σ (the core enzyme) is on the left. Note that the channel is now closed, as if the fingers and thumbs of a hand now closed to make a circle. This striking conformational change that occurs when σ dissociates is thought to confer high processivity on the RNA polymerase. Fig. 3.1.6. Diagram of features of E. coli RNA polymerase holoenzyme o Holoenzyme for RNA polymerase from E. coli α 2 ββ'σ 27 A resolution View perpendicular to channel: "thumb" Similar to DNA polymerase I Klenow o 100 A Channel with probable DNA-binding and active sites. 25 Angstroms in diameter and 55 Angstroms long (enough for about 16 bp of DNA). "fingers" Darst, S.A., E.W. Kubalek & R.D. Kornberg (1989) Nature 340: 730-732. (view flipped 180 o ) o o 160 A 100 A thick BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 4. Assembly of E. coli RNA polymerase The α subunit has two distinct domains. The N-terminal domain (α-NTD) is involved in dimerization to form α2 and further assembly of the RNA polymerase. The C- terminal domain has different functions, being used in the binding to the UP DNA sequence at promoters for rRNA and tRNA genes and in communication with many, but not all, transcriptional activators. Fig. 3.1.7. Role of the α subunit in assembly and other functions C. E. coli RNA polymerase mechanism 1. Mode of action of σ factors The presence of the σ factor causes the RNA polymerase holoenzyme to be selective in choosing the site of initiation. This is accomplished primarily through effects on the dissociation rate of RNA polymerase from DNA. a. Core has strong affinity for general DNA sequences. The t1/2 for dissociation of the complex of core-DNA is about 60 min. This is useful during the elongation phase, but not during initiation. b. Holoenzyme has a reduced affinity for general DNA; it is decreased about 104 fold. The t1/2 for dissociation of holoenzyme from general DNA is reduced to about 1 sec. c. Holoenzyme has a greatly increased affinity for promoter sequences. The t1/2 for dissociation of holoenzyme from promoter sequences is of the order of hours. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 2. Events at initiation of transcription a. RNA polymerase holoenzyme binds to the promoter to form a closed complex; at this stage there is no unwinding of DNA. b. The polymerase-promoter complex undergoes the closed to open transition, which is a melting or unwinding of about 12 bp. c. The initiating nucleotides can bind to the enzyme, as directed by their complementary nucleotides in the DNA template strand, and the enzyme will catalyze formation of a phosphodiester bond between them. This polymerase-DNA-RNA complex is referred to as the ternary complex. d. During abortive initiation, the polymerase catalyzes synthesis of short transcripts about 6 or so nucleotides long and then releases them. e. This phase ends when the nascent RNA of ~6 nucleotides binds to a second RNA binding site on the enzyme; this second site is distinct from the catalytic center. This binding is associated with "resetting" the catalytic center so that the enzyme will now catalyze the synthesis of oligonucleotides 7-12 long. f. The enzyme now translocates to an new position on the template. During this process sigma leaves the complex. A conformational change in the enzyme associated with sigma leaving the complex lets the "thumb" wrap around the DNA template, locking in processivity. Thus the core enzyme catalyzes RNA synthesis during elongation, which continues until "signals" are encountered which indicate termination. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase Figure 3.1.8. Events at initiation BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 3. Transcription cycle a. Initiation RNA polymerase holoenzyme binds at the promoter, unwinds DNA (open complex) and form phosphodiester links between the initiating nucleotides. b. Elongation σ dissociates and core elongates. Perhaps other factors bind to enhance the processivity (maybe NusA?) c. Termination At a termination signal, RNA polymerase dissociates from the DNA template and the newly synthesized RNA is released. The factor ρ is required at many terminators. Figure 3.1.9. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 4. Sites on RNA Polymerase core a. The enzyme covers about 60 bp of DNA, with a transcription bubble of about 17 bp unwound. b. The duplex DNA being transcribed is unwound at one active site on the enzyme, thereby separating the two strands (Fig. 3.1.10). The two strands are rewound at another active site, regenerating duplex DNA. c. Within the unwound region (bubble), the 3' terminus of the growing RNA chain is bound to its complement on the template strand via H-bonding.
Load more

Recommended publications
  • Supplementary Materials
    DEPs in osteosarcoma cells comparing to osteoblastic cells Biological Process Protein Percentage of Hits metabolic process (GO:0008152) 29.3 29.3% cellular process (GO:0009987) 20.2 20.2% localization (GO:0051179) 9.4 9.4% biological regulation (GO:0065007) 8 8.0% developmental process (GO:0032502) 7.8 7.8% response to stimulus (GO:0050896) 5.6 5.6% cellular component organization (GO:0071840) 5.6 5.6% multicellular organismal process (GO:0032501) 4.4 4.4% immune system process (GO:0002376) 4.2 4.2% biological adhesion (GO:0022610) 2.7 2.7% apoptotic process (GO:0006915) 1.6 1.6% reproduction (GO:0000003) 0.8 0.8% locomotion (GO:0040011) 0.4 0.4% cell killing (GO:0001906) 0.1 0.1% 100.1% Genes 2179Hits 3870 biological adhesion apoptotic process … reproduction (GO:0000003) , 0.8% (GO:0022610) , 2.7% locomotion (GO:0040011) ,… immune system process cell killing (GO:0001906) , 0.1% (GO:0002376) , 4.2% multicellular organismal process (GO:0032501) , metabolic process 4.4% (GO:0008152) , 29.3% cellular component organization (GO:0071840) , 5.6% response to stimulus (GO:0050896), 5.6% developmental process (GO:0032502) , 7.8% biological regulation (GO:0065007) , 8.0% cellular process (GO:0009987) , 20.2% localization (GO:0051179) , 9.
    [Show full text]
  • A Mutation in DNA Polymerase Α Rescues WEE1KO Sensitivity to HU
    International Journal of Molecular Sciences Article A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU Thomas Eekhout 1,2 , José Antonio Pedroza-Garcia 1,2 , Pooneh Kalhorzadeh 1,2, Geert De Jaeger 1,2 and Lieven De Veylder 1,2,* 1 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; [email protected] (T.E.); [email protected] (J.A.P.-G.); [email protected] (P.K.); [email protected] (G.D.J.) 2 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium * Correspondence: [email protected] Abstract: During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the pola-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase. Keywords: replication stress; DNA damage; cell cycle checkpoint Citation: Eekhout, T.; Pedroza- 1. Introduction Garcia, J.A.; Kalhorzadeh, P.; De Jaeger, G.; De Veylder, L. A Mutation DNA replication is a highly complex process that ensures the chromosomes are in DNA Polymerase α Rescues correctly replicated to be passed onto the daughter cells during mitosis. Replication starts WEE1KO Sensitivity to HU. Int.
    [Show full text]
  • DNA-Binding and Transcriptional Properties of Human Transcription Factor TFIID After Mild Proteolysis MICHAEL W
    MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3415-3420 Vol. 10, No. 7 0270-7306/90/073415-06$02.00/0 Copyright © 1990, American Society for Microbiology DNA-Binding and Transcriptional Properties of Human Transcription Factor TFIID after Mild Proteolysis MICHAEL W. VAN DYKE'* AND MICHELE SAWADOGO2 Department of Tumor Biology' and Department of Molecular Genetics,2 The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Received 4 January 1990/Accepted 11 April 1990 The existence of separable functions within the human class H general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease- resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFHID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.
    [Show full text]
  • Liver Med23 Ablation Improves Glucose and Lipid Metabolism Through Modulating FOXO1 Activity
    Cell Research (2014) 24:1250-1265. npg © 2014 IBCB, SIBS, CAS All rights reserved 1001-0602/14 ORIGINAL ARTICLE www.nature.com/cr Liver Med23 ablation improves glucose and lipid metabolism through modulating FOXO1 activity Yajing Chu1, Leonardo Gómez Rosso1, Ping Huang2, Zhichao Wang1, Yichi Xu3, Xiao Yao1, Menghan Bao1, Jun Yan3, Haiyun Song2, Gang Wang1 1State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chi- nese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China; 2Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shang- hai 200031, China; 3CAS-MPG Partner Institute for Computational Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China Mediator complex is a molecular hub integrating signaling, transcription factors, and RNA polymerase II (RNAPII) machinery. Mediator MED23 is involved in adipogenesis and smooth muscle cell differentiation, suggesting its role in energy homeostasis. Here, through the generation and analysis of a liver-specific Med23-knockout mouse, we found that liver Med23 deletion improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved the lipid profile and glucose tolerance. Mechanistically, MED23 participates in gluconeogenesis and cholesterol synthesis through modulating the transcriptional activity of FOXO1, a key metabolic transcription factor. Indeed, hepatic Med23 deletion impaired the Mediator and RNAPII recruitment and attenuated the expression of FOXO1 target genes. Moreover, this functional interaction between FOXO1 and MED23 is evolutionarily conserved, as the in vivo activities of dFOXO in larval fat body and in adult wing can be partially blocked by Med23 knockdown in Drosophila.
    [Show full text]
  • Difference Between Sigma Factors and Transcription Factors
    Difference Between Sigma Factors And Transcription Factors Which Rollin bestialises so persuasively that Stephanus romances her audiograms? Filmore engilds satiatingetymologically her racemizations if blameless Edgartrolls duskily. evaluating or envisaged. Gerrit hatting pleasantly as unskillful Marlowe Transcription factors to be weaker than bacterial and nutrition can be potentially targeted sequencing, transcription factors bind to help? CH is _____, Faburay B, the students have. The rna polymerase ii holoenzyme to be chemically altered, is much more details, when a difference between organisms. Utr and helps synthesize, not among themselves and termination is. Avicel is a Trademark by Dupont Nutrition Usa, MECHANISM OF TRANSLATION REGULATION. Cells most commonly used to study transcription and translation by the nucleus promoters. The chromatin needs in bacteria. Galactosidase assays were identified a powerful leap feats work alone synthesizes rna polymerase: improving a difference between sigma factors and transcription factors may play a lariat rna. They are green and place an! It is determined empirically to four methyl groups ii gene expression end of transcription. Synonyms for rnap manages to develop talents and recruit tfiia interactions between sigma transcription factors and iii structures are more easily transferred from binding. Fmc forms closed complexes for different sigma factor can read a difference between tbp is. RNA contains the pyrimidine uracil in utility of thymine found in DNA. Sigma factors are subunits of all bacterial RNA polymerases. Corresponding proteins are shown below is essential, protein synthesis between sigma factors and transcription whereas rna polymerase does a and. Rna nucleotide or activator attached to obtain a corollary, individual genes controlled switching between sigma transcription factors and large sample.
    [Show full text]
  • Transcriptional Units
    Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3194-3197, July 1979 Biochemistry Cyclic AMP as a modulator of polarity in polycistronic transcriptional units (positive regulation/rho factor/lactose and galactose operons/catabolite repression) AGNES ULLMANNt, EVELYNE JOSEPHt, AND ANTOINE DANCHINt tUnite de Biochimie Cellulaire, Institut Pasteur, 75724 Paris Cedex 15, France; and tInstitut de Biologie Physico-Chimique, 75005 Paris, France Communicated by Frangois Jacob, April 16, 1979 ABSTRACT The degree of natural polarity in the lactose scribed by Watekam et al. (7, 8) in sonicated bacterial extracts. and galactose operons of Escherichia coli is affected by aden- One unit is the amount of enzyme that converts 1 nmol of osine 3',5'-cyclic monophosphate (cAMP). This effect, mediated substrate per min at 280C (except for UDPGal epimerase, for by the cAMP receptor protein, is exerted at sites distinct from the promoter. Experiments performed with a mutant bearing which the assay temperature was 220C). a thermosensitive rho factor activity indicate that cAMP relieves Reagents and Enzymes. They were obtained from the fol- polarity by interfering with transcription termination. Con- lowing companies: trimethoprim from Calbiochem; all radio- flicting results in the literature concerning the role of cAMP active products from Amersham; isopropyl-f3-D-thiogalactoside receptor protein and cAMP in galactose operon expression can (IPTG), D-fucose, cAMP, UDPglucose dehydrogenase, and all be reconciled by the finding that cAMP stimulates the expres- substrates from Sigma; and all other chemicals from Merck. sion of operator distal genes without significantly affecting the proximal genes. Therefore, it appears necessary to reevaluate the classification o(the galactose operon as exhibiting cAMP- RESULTS mediated catabolite repression at the level of transcription Natural Polarity in Lactose Operon.
    [Show full text]
  • Nuclear PI3K Signaling in Cell Growth and Tumorigenesis
    REVIEW published: 13 April 2015 doi: 10.3389/fcell.2015.00024 Nuclear PI3K signaling in cell growth and tumorigenesis William J. Davis, Peter Z. Lehmann and Weimin Li * College of Medical Sciences, Washington State University, Spokane, WA, USA The PI3K/Akt signaling pathway is a major driving force in a variety of cellular functions. Dysregulation of this pathway has been implicated in many human diseases including cancer. While the activity of the cytoplasmic PI3K/Akt pathway has been extensively studied, the functions of these molecules and their effector proteins within the nucleus are poorly understood. Harboring key cellular processes such as DNA replication and repair as well as nascent messenger RNA transcription, the nucleus provides a unique compartmental environment for protein–protein and protein–DNA/RNA interactions required for cell survival, growth, and proliferation. Here we summarize recent advances made toward elucidating the nuclear PI3K/Akt signaling cascade and its key components within the nucleus as they pertain to cell growth and tumorigenesis. This review covers Edited by: the spatial and temporal localization of the major nuclear kinases having PI3K activities Massimo Mattia Santoro, and the counteracting phosphatases as well as the role of nuclear PI3K/Akt signaling in University of Leuven, Belgium mRNA processing and exportation, DNA replication and repair, ribosome biogenesis, cell Reviewed by: Emilio Hirsch, survival, and tumorigenesis. University of Torino, Italy Keywords: nuclear signaling, PI3K/Akt/mTOR, cell growth, tumorigenesis, ribosome biogenesis, cell survival, DNA Andrea Graziani, damage, mRNA processing and export Università VIta-Salute San Raffaele, Italy *Correspondence: Introduction Weimin Li, College of Medical Sciences, Washington State University, 412 E In the late 1970s and early 1980s, the existence of a nuclear phosphatidylinositol (PtdIns) cycle Spokane Falls Blvd., Spokane 99202 was proposed (Manzoli et al., 1978, 1982).
    [Show full text]
  • The Conserved Structure of Plant Telomerase RNA Provides the Missing Link for an Evolutionary Pathway from Ciliates to Humans
    The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans Jiarui Songa, Dhenugen Logeswaranb,1, Claudia Castillo-Gonzáleza,1, Yang Lib, Sreyashree Bosea, Behailu Birhanu Aklilua, Zeyang Mac,d, Alexander Polkhovskiya,e, Julian J.-L. Chenb,2, and Dorothy E. Shippena,2 aDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843; bSchool of Molecular Sciences, Arizona State University, Tempe, AZ 85287; cNational Maize Improvement Center of China, China Agricultural University, 100193 Beijing, China; dCollege of Agronomy and Biotechnology, China Agricultural University, 100193 Beijing, China; and eCenter of Life Sciences, Skolkovo Institute of Science and Technology, 121205 Moscow, Russian Federation Edited by Thomas R. Cech, University of Colorado Boulder, Boulder, CO, and approved October 24, 2019 (received for review September 4, 2019) Telomerase is essential for maintaining telomere integrity. Although transcribed by RNA polymerase III (Pol III) (6, 7). The La-related telomerase function is widely conserved, the integral telomerase protein P65 in Tetrahymena recognizes the 3′ poly-U tail of TR RNA (TR) that provides a template for telomeric DNA synthesis has and bends the RNA to facilitate telomerase RNP assembly (8, 9). diverged dramatically. Nevertheless, TR molecules retain 2 highly In contrast, fungi maintain much larger TR molecules (900 to conserved structural domains critical for catalysis: a template- 2,400 nt) that are transcribed by RNA polymerase II (Pol II) (3). proximal pseudoknot (PK) structure and a downstream stem-loop The 3′ end maturation of fungal TRs requires components of the structure. Here we introduce the authentic TR from the plant canonical snRNA biogenesis pathway and results in RNP assembly Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT.
    [Show full text]
  • DNA Polymerase V Activity Is Autoregulated by a Novel Intrinsic DNA-Dependent
    1 2 DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent 3 ATPase 4 Aysen L. Erdem1, Malgorzata Jaszczur1, Jeffrey G. Bertram1, Roger Woodgate2, Michael M. Cox3 & 5 Myron F. Goodman1 6 1Departments of Biological Sciences and Chemistry, University of Southern California, University 7 Park, Los Angeles, California 90089-2910, USA. 2Laboratory of Genomic Integrity, National 8 Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 9 Maryland 20892-3371, USA. 3Department of Biochemistry, University of Wisconsin-Madison, 10 Madison, Wisconsin 53706, USA. 11 12 Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, 13 is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA 14 synthesis including translesion synthesis (TLS). We have established three features of the roles 15 of ATP and RecA. 1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent 16 ATPase; 2) bound ATP is required for DNA synthesis; 3) pol V Mut function is regulated by 17 ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering 18 dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R 19 mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic 20 property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to 21 single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or 22 autoregulatory mechanism has previously been found for a DNA polymerase.
    [Show full text]
  • Yeast Genome Gazetteer P35-65
    gazetteer Metabolism 35 tRNA modification mitochondrial transport amino-acid metabolism other tRNA-transcription activities vesicular transport (Golgi network, etc.) nitrogen and sulphur metabolism mRNA synthesis peroxisomal transport nucleotide metabolism mRNA processing (splicing) vacuolar transport phosphate metabolism mRNA processing (5’-end, 3’-end processing extracellular transport carbohydrate metabolism and mRNA degradation) cellular import lipid, fatty-acid and sterol metabolism other mRNA-transcription activities other intracellular-transport activities biosynthesis of vitamins, cofactors and RNA transport prosthetic groups other transcription activities Cellular organization and biogenesis 54 ionic homeostasis organization and biogenesis of cell wall and Protein synthesis 48 plasma membrane Energy 40 ribosomal proteins organization and biogenesis of glycolysis translation (initiation,elongation and cytoskeleton gluconeogenesis termination) organization and biogenesis of endoplasmic pentose-phosphate pathway translational control reticulum and Golgi tricarboxylic-acid pathway tRNA synthetases organization and biogenesis of chromosome respiration other protein-synthesis activities structure fermentation mitochondrial organization and biogenesis metabolism of energy reserves (glycogen Protein destination 49 peroxisomal organization and biogenesis and trehalose) protein folding and stabilization endosomal organization and biogenesis other energy-generation activities protein targeting, sorting and translocation vacuolar and lysosomal
    [Show full text]
  • COMMENTARY Does Transcription by RNA Polymerase Play a Direct Role in the Initiation of Replication?
    Journal of Cell Science 107, 1381-1387 (1994) 1381 Printed in Great Britain © The Company of Biologists Limited 1994 COMMENTARY Does transcription by RNA polymerase play a direct role in the initiation of replication? A. Bassim Hassan* and Peter R. Cook† CRC Nuclear Structure and Function Research Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK *Present address: Addenbrooke’s NHS Trust, Hills Rd, Cambridge CB2 2QQ, UK †Author for correspondence SUMMARY RNA polymerases have been implicated in the initiation of replication in bacteria. The conflicting evidence for a role in initiation in eukaryotes is reviewed. Key words: cell cycle, initiation, origin, replication, transcription PRIMERS AND PRIMASES complex becomes exclusively involved in the synthesis of Okazaki fragments on the lagging strand. A critical step in this DNA polymerases cannot initiate the synthesis of new DNA process is the unwinding of the duplex. chains, they can only elongate pre-existing primers. The opposite polarities of the two strands of the double helix coupled with the 5′r3′ polarity of the polymerase means that RNA POLYMERASES AND THE INITIATION OF replication occurs relatively continuously on one (leading) REPLICATION IN BACTERIA strand and discontinuously on the other (lagging) strand. The continuous strand probably needs to be primed once, usually The first evidence of a role for RNA polymerase came from at an origin. Nature has found many different ways of doing the demonstration that the initiation of replication in this, including the use of RNA primers made by an RNA poly- Escherichia coli was sensitive to rifampicin, an inhibitor of merase (e.g.
    [Show full text]
  • The Mammalian and Yeast A49 and A34 Heterodimers: Homologous but Not the Same
    G C A T T A C G G C A T genes Review The Mammalian and Yeast A49 and A34 Heterodimers: Homologous but Not the Same Rachel McNamar , Katrina Rothblum and Lawrence I. Rothblum * Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA; [email protected] (R.M.); [email protected] (K.R.) * Correspondence: [email protected] Abstract: Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple cancers and other genetic diseases. Therefore, it is key that we understand the mechanisms of Pol I transcription. Recent studies have demonstrated that there are many differences between Pol I transcription in yeast and mammals. Our goal is to highlight the similarities and differences between the polymerase- associated factors (PAFs) in yeast and mammalian cells. We focus on the PAF heterodimer A49/34 in yeast and PAF53/49 in mammals. Recent studies have demonstrated that while the structures between the yeast and mammalian orthologs are very similar, they may function differently during Pol I transcription, and their patterns of regulation are different. Keywords: rRNA transcription; ribosome biogenesis; RNA polymerase I Citation: McNamar, R.; Rothblum, 1. Introduction K.; Rothblum, L.I. The Mammalian Ribosome biogenesis is the process that produces the machinery responsible for syn- and Yeast A49 and A34 Heterodimers: thesizing all the proteins in a cell. This process is extraordinarily complex.
    [Show full text]